Investigating the effect of antimicrobials

In a nutshell

Antibiotics and antiseptics are antimicrobials that prevent bacterial growth. The impacts of different antibiotics and antiseptics can be measured using zones of inhibition. Different antimicrobial-soaked discs are placed on agar plates. If they kill the bacteria there will be a clear zone of inhibition surrounding the disc. The zone of inhibition can be measured and compared to determine which antimicrobials are the most effective.

Equipment list

The following equipment can be used to investigate the different impacts of antibiotics and antiseptics on bacterial growth.

Equipment	Use
A nutrient agar plate	This will be the growth medium for your bacterial culture.
A bunsen burner	To sterilise equipment.
A heatproof mat	To place the Bunsen burner on.
Filter paper discs	To soak the antiseptic and antibiotics in.
Antimicrobials	This will depend on the practical you are doing. You may be provided with mouthwash, TCP, antiseptic cream or other antimicrobials.
Water	To soak the paper disc in as a control.
Bacterial culture	This will be the bacteria you are trying to inhibit the growth of. Usually, this will be E. coli.
Disposable plastic pipettes	To transfer solutions.
Glass spreader	To spread the bacteria on the nutrient agar plate.
Disinfectant spray	To clean the work surfaces before and after the practical.
'Discard beaker' of disinfectant	To place the used contaminated equipment in.
Forceps	To transfer the filter paper discs.
Tape	To seal the agar plate.
Hand wash	To wash your hands before and after the practical.
Wax pencil or permanent marker	To write on the agar plate.
Incubator	To incubate the bacteria at $25\ \degree C$ .

Safety precautions

When carrying out experiments, it is very important to consider the safety precautions so nobody gets hurt.

Hazard	Risk	Safety measure
Bacteria	Bacteria is infectious.	Take care when handling the bacterial culture and use aseptic techniques to avoid contamination. Also, ensure your work spaces and hands are thoroughly cleaned before and after handling the practical.
Bunsen burner	You may burn yourself.	Be careful around the Bunsen burner and tie hair up.

Experiment 1: Investigate the effect of antiseptics or antibiotics on bacterial growth.

The impact of antiseptics and antibiotics on bacterial growth can be measured using agar plates and measuring the zones of inhibition.

Experimental variables

Every experiment will have an independent, dependent and constant variable. The independent variable is the variable that you change. The dependent variable is the variable that depends on the independent variable; therefore, it is the one you measure. The control variable is what is kept the same during the experiments.

Independent variable	$Type\space of\space antimicrobial.$
Dependent variable	$Diameter\space of\space the\space zone\space of\space inhibition.$
Control variables	$Incubation\space temperature,\space type\space of\space bacteria,\space amount\space of\space bacterial\space culture,\space\\ type\space of\space agar,\space size\space disc,\space concentration\space of\space antimicrobial\space and\space\\ incubation\space time.$

Method

The following method is used to investigate the effect of antiseptics or antibiotics on bacterial growth.

1.	Spray the bench with the disinfectant spray and wipe with paper towels.
2.	Set up the heatproof mat with the Bunsen burner in the middle of your bench.
3.	Set the Bunsen burner to a yellow flame.
4.	Wash your hands using the antibacterial hand wash.
5.	Turn the agar plate upside down and using the pencil or permanent marker, divide the plate into four equally sized sections. Number the sections $1, 2, 3$ and $4$ at the outer edge. Draw a dot in the middle of each section. Around the edge of the plate write your initials, the date and the name of the bacteria you are using. Note: You must always write on the bottom of the agar plate as they are stored upside down and if the lid becomes separated from the plate, you will still know what is growing on the plate. *Note:* You must always write the date and the type of bacteria you are working on so other scientists can take the necessary precautions.
6.	Turn the Bunsen burner flame to blue.
7.	Remove the lid of the bacterial culture vessel and flame the neck of the bottle in the Bunsen burner. *Note:* Keep the lid in your hand and do not put it on the bench.
8.	Use a disposable pipette to collect $1\ ml$ of the bacterial culture.
9.	Flame the neck of the bacterial culture vessel again and put the lid back on it.
10.	Slightly lift the lid of the agar plate and pipette the bacteria onto the agar. Quickly replace the lid. *Note:* The agar plate should not be fully opened to prevent contamination.
11.	Immediately place the pipette in the beaker containing the disinfectant.
12.	Turn the Bunsen burner flame back to yellow.
13.	Take the glass spreader and dip it into the ethanol.
14.	Pass the glass spreader through the Bunsen burner. *Note:* Hold the glass spreader at an angle so nothing drips down onto your skin.
15.	Remove the glass spreader from the flame and allow it to cool for $20\ seconds$ . *Note:* You must allow the glass spreader to cool otherwise it will be too hot and it will kill the bacteria on the plate.
16.	Partially lift the lid of the agar plate and use the glass spreader to spread the bacteria around the nutrient agar.
17.	Shut the lid of the agar plate and place the glass spreader into the beaker containing the disinfectant.
18.	Soak or spread the different filter paper discs with the different antiseptics. *Note:* Leave one soaked in water as a control.
19.	Partially lift the lid of the agar plate and, using forceps, place one disc on each dot in each sector of the agar plate. *Note:* Make sure you write down which antimicrobial is in which section.
20.	Put the lid back on the agar plate and secure it in place with two small pieces of tape. *Note:* Do not tape the whole lid as this will create anaerobic conditions. The E. coli bacteria will not grow and anaerobic bacteria will grow instead (these are harmful bacteria).
21.	Incubate the plate for $48\ hours$ at $25\ \degree C$ .

Analysis

This is how you would use your data to form conclusions.

1.	Measure the diameter of the clear zone around each disc by placing the ruler across the centre of the disc. *Note:* Do not open the agar plate to measure the clear zones. This can be achieved by looking down at the agar plate.
2.	Measure again at $90\degree$ to the first measurement so that the mean diameter can be calculated.
3.	Calculate the mean diameter using the equation below. $mean=\frac{diameter\space 1 + diameter \space 2}{2}$
4.	To get more accurate results, you may need to calculate the area of the zone of inhibition. $area=\pi r^2$

Biology; Biology practicals; KS4 Year 10; Investigating the effect of antimicrobials

Conclusion

The antimicrobials will diffuse out of the filter paper disc and prevent the growth of the bacteria. The larger the clear zone around a disc, the more effective the antibiotic is against the bacteria. Using your area, you can work out which antimicrobials are the most effective. The water is used as a control which means that any difference between the growth of the bacteria around the paper discs is because of the antimicrobials and not the effect of the paper.

Evaluation

After completing your experiment you must comment on the quality of your data and suggest improvements to the method you have used.

A common source of error is that the agar plate was taped incorrectly. If this is the case, the agar plate is considered hazardous as the bacteria that is growing on the plate is not E. coli. This plate would not be included in the results.

If there is no bacteria growing on the plate, it is likely to be a result of a human error. If the inoculating loop is not left to cool enough before the bacterial sample is taken, the bacteria may be killed. Therefore, no bacteria will grow.

The calculations are also a common source of error. Any mistakes when measuring the zone of inhibition will impact your final results. Therefore, you should use a ruler with clear markings and be careful when reading the ruler.